Initiation of human DNA replication entails a highly regulated assembly of polymerase complexes along with chaperone proteins and other enzymes. The polymerase alpha with its associated primase activity is the only polymerase involved in this initiation process. Our research is concentrated on understanding the regulation and mechanism of the human DNA polymerase alpha - primase complex. This enzyme complex is composed of four proteins, a 180 kDa phospho-glycoprotein containing the DNA polymerase activity, a 70 kDa phosphoprotein with no known function, and two smaller subunits of 49 and 58 kDa containing the primase activity. Current research is focused on the enzymatic mechanism and regulation of the two primase subunits. The cDNA for the two human DNA primase subunits (Hp49 and Hp58) were isolated and sequenced. The Hp49 and Hp58 subunits were overexpressed separately and together in E. coli and proteins purified. The recombinant human primase subunits are being used to study protein-protein interactions within the DNA polymerase alpha- primase complex as well as to identify other replication or repair proteins which may interact with these primase subunits. Deletions of the Hp58 primase subunit were also constructed, overexpressed and analyzed for its role in primer formation. Deletion constructs of the Hp58 subunit were made to investigate its role in primer formation. Analysis of the complexes formed between the deleted Hp58 constructs and the native Hp49 subunit revealed that protein-protein contacts between the two subunits are made over several domains of the Hp58 subunit. Of four primase complexes containing different Hp58 deletions only one complex was able to support initiation as measured by the formation of dinucleotides.